Zea mays L. or corn is a major worldwide cereal crop. In the continental United States alone, an estimated 82 million acres of corn is planted yearly. A survey of the U.S. corn germplasm base conducted in 1979 accounting for 1.3 billion pounds of seed corn, determined that corn inbred B73 was used in the production of approximately 178.5 million pounds of corn hybrid seed. This amount reflects approximately 14% of the total corn seed required for the 1980 planting of 82 million acres of corn in the United States. B73 was used to produce more hybrid corn seed than any other corn inbred line in the 1979 survey. As the major inbred corn line used in hybrid corn seed production, B73 is a commercially important corn inbred. Successful efforts to improve B73 are likely to have a major impact on the commercial corn seed business.
Current methods for improving inbred corn lines are time consuming, labor intensive, and risky. Thus, methods that reduce any of these problems would represent an advance in the cereal breeding arts. One method for inducing changes in an inbred corn seed line is accomplished by exposing inbred seed corn to a mutagen, which may be ionizing radiation or a mutagenic chemical, growing the seed out, and screening the corn plants for a desired characteristic such as decreased height or pathogen resistance. Seed from plants displaying the desired characteristics are then replanted, rescreened and the seed from plants passing the screen are retained. After several years and many grow-outs of the seed, a sufficient seed stock of the inbred displaying the desired characteristic is accumulated for commercial breeding.
Several problems attend the method of obtaining a modified inbred corn seed line as described above. Inbred corn lines are highly homozygous, or pure-bred. Such inbred lines frequently display low vigor, and as a result frequently produce few seed. If seed production is too low, successful large-scale production of seed derived from a single plant of a modified inbred line may not be possible at all. Even if the modified inbred is vigorous and produces numbers of fertile seed, several years and many acres of land are required to produce enough seeds to make commercial breeding possible.
Other approaches to the propagation of desirable modified inbreds have also been suggested and rely upon plant tissue culture techniques. In some of these approaches, cell cultures are subjected to mutagenic ionizing radiation. The desirable characteristic is selected or screened in cell culture. Cells having the desirable characteristic are propagated in culture and ultimately regenerated to form seed-bearing plants (see e.g., Bottino P. J., "The Potential of Genetic Manipulation of Plant Cell Cultures for Plant Breeding," Radiation Botany, 15:1-16 (1975).
The feasibility of using selection techniques on callus cultures of certain non-commercial corn inbreds and the regeneration of plants producing seed that carry the characteristic selected in tissue culture has been demonstrated by Hibberd et al. (see, "Selection and Characterization of a Feedback-Insensitive Tissue Culture of Maize, " Planta, 148:183-187 (1980).)
Prior to the development of the embroygenic callus line of corn inbred B73 of the instant invention, no friable embryogenic callus culture or embryogenic cell suspension culture of a commercially significant inbred corn line has been described. With respect to embryogenic callus lines of noncommercial corn inbreds, C. E. Green has described a friable callus of corn inbred A188 capable of regenerating plants by somatic embroygenesis (see, Green, C. E., "Somatic Embroygenesis and Plant Regeneration From the Friable Callus of Zea mas L." in Proc. 5th Int'l. Cong. Plant Tissues and Cells, Akio Jujiware ed., Japanese Association of Plant Tissue Culture, pub. Tokyo, Japan, 1982). Embryogenic friable A188 callus according to Green can be derived from spontaneous sectors of organogenic callus.
Green's embryogenic callus appears undifferentiated, highly friable, has little organization on visual inspection and is fast growing, requiring sub-culturing at least every two weeks. The embryogenic callus culture described by Green develops to the coleoptilar stage on MS or N6 medium with 2% sucrose and 0.5 to 1.0 mg/l 2,4-D. Normal development to the mature embryo stage is possible if the embryos are transferred to N6 medium with 6% sucrose and no 2,4-D. The mature embryo can then be induced to germinate on MS or N6 medium containing 2% sucrose without hormones.
Green also described suspension cultures of the embryogenic lines of A188 and disclosed that such suspension cultures can be maintained in MS medium containing 1 mg/1 2,4-D with 2% sucrose. When aliquots of the suspension cultures are plated onto solidified MS medium containing 2% sucrose, a callus forms from the plated suspensions and embryos form on the callus surface. These embryos are transferred to MS or N6 medium with 6% sucrose for further development. After 2 weeks on medium with 6% sucrose these are transferred to low sucrose, hormone-free medium for embryo generation. A188 plants may be routinely regenerated from these embryos.
A non-regenerating cell line of corn inbred B73 is known (see Potrykus et al, Theoretical Applied Genetics, 54:209, 1979). This cell line in suspension culture was established from a primary culture of protoplasts from plant tissues of the B73 inbred. The cells are characterized as growing well in suspension culture and easily maintainable on agar medium. Furthermore, the cells can be enzymatically turned into protoplasts, which in turn regenerate walls and become proliferating cells again. Significantly, the cells of this line have never been induced to regenerate plants and moreover display ploidy abnormalities, a characteristic well known in other non-regenerating diploid plant cell lines. (See M. G. Meadows, "Characterization of Cells and Protoplasts of the B73 Maize Cell Line " Plant Science Letters, 28:337-348(1982/83). Other attempts to achieve plant regeneration from established B73 callus have been similarly unsuccessful. (See Bartkowiak, E., "Tissue Culture of Maize Plantlet Regeneration From Scutella Callus," Genetica Polonica, Vol. 23:93-101 (1982)).
The ability to culture somatic cells of inbred plants in vitro enables the plant breeder to apply the techniques of microbial genetics to specific breeding problems in crop plants. Included in the genetic approaches that have application to plant breeding are: selection of mutations in cell cultures, studying host-pathogen interrelationships, transfer of genetic information into cells through uptake of exogenous heterologous (see Szoka et al., U.S. Pat. No. 4,394,448) or homologous (see IPRI European Patent Application No. 90033A) DNA. Furthermore, somatic cell culture enables the plant breeder to develop, select and propagate somaclonal variants having novel or useful agronomic characteristics.
The major obstacle to the application of these techniques to commercial corn inbred B73 and some other commercial inbreds has, until now, been the inability to provide a tissue culture or cell suspension that can regenerate whole plants. The embryogenic corn callus line and corn cell line derived therefrom provided by the instant invention makes it possible to apply somatic cell genetic techniques to a major commercial corn line for the first time. The particular uses to which a cereal, and particularly a corn cell line such as the one described herein, can be put are outlined in numerous articles (see e.g., Bottino, P. J. "Potential of Genetic Manipulation in Plant Cell Culture," Radiation Botany, Vol 15:1-16 (1975) and Vasil, I. K., "Plant Cell Culture and Somatic Cell Genetics of Cereals and Grasses," Plant Improvement and Somatic Cell Genetics, Vasil Ed., Academic Press, N.Y., 1st Ed., (1983) and "Tissue Culture in the Production of Novel Disease-Resistant Crop Plants," Biol. Rev., 54:329-345 (1975)).
These uses include selection at the cellular level of mutations for resistance to toxic substances, such as herbicides and substances produced by plant pathogens. Since the selections are carried out at the cellular level, it is likely that whole plants regenerated from the cells will show the selected characteristic. It is significant that such a system would allow plant breeders to select (or screen) for the desired characteristic from among thousands of cells in a single culture dish or flask, whereas a large field plot would be required to select or screen a corresponding number of seed grown plants according to traditional plant breeding methods.
It has, for example, been demonstrated that corn plants derived from Black Mexican Sweet corn, a non-commercial cultivar, can be regenerated from tissue cultures that have been selected for resistance to lysine and threonine and that tissues of the regenerated plant likewise produce corn plant tissue that is resistant to lysine and threonine. (See Hibberd, K. A. et al., "Selection and Characterization of a Feedback Insensitive Tissue Culture of Maize," Planta, 148:183-187 (1980).)
The utility of such selections carried out in callus cultures has also been demonstrated by the regeneration from callus culture of corn plants resistant to southern corn leaf blight, a plant disease caused by a pathotoxin produced by the plant pathogen Helminthosporium maydis race T. By exposing corn callus to the pathotoxin produced by the plant pathogen, resistant callus was selected. Significantly, the progeny of certain resistant plants were also resistant to the toxin and plant pathogen. Thus callus culture of a corn cell line is demonstrably effective in the development of pathogen resistant corn seed. (See Gengenbach et al., "Inheritance of Selected Pathotoxin Resistance in Maize Plants Regenerated From Cell Cultures," Proc. Nat'l. Acad. Sci. USA, 74:5113-5117 (1977)).